A number of neural crest derivatives have been shown, by manipulation of developmental signals in cell culture, to display remarkable plasticity in the range of phenotypes they can assume. It is proposed to study the hierarchy and timing of these choices in the sympathoadrenal lineage during normal development in vivo, to characterize the cellular and hormonal signals which control these differentiation decisions, and to determine the role of these signals in vivo. I. A protein secreted by heart cells can convert postmitotic adrenergic neurons into cholinergic neurons. This molecule has been purified 10000-fold from serum-free conditioned medium. The purity of this preparation will be assessed on 2-dimensional gels and by N-terminal analysis. If homogeneous, its amino acid sequence will be determined. Monoclonal antibodies against this protein are being prepared by in vitro immunization; polyclonal antisera will also be produced for experiments to determine the role this factor plays in neuronal development in vivo. A similar cholinergic factor is found in rat serum, where it appears with the same developmental time course as the cholinergic differentiation of the sympathetic innervation of the sweat glands. The properties of this developmentally controlled, in vivo cholinergic factor will be compared to those of the better characterized protein in conditioned medium. II. Previous work on the interconversions of adrenal chromaffin cells, small intensely fluorescent (SIF) cells and sympathetic neurons led to the hypothesis that SIF cells are the central intermediate cell type of the sympathoadrenal lineage and are the precursors for much of the autonomic nervous system. This idea will be tested by modulating the number of SIF cells during development and observing the consequences on the other derivatives of the lineage. Glucocorticoid and NGF levels in pregnant rats will be modified by injections so as to increase the number of SIF cells. Conversely, it is proposed to selectively kill SIF cells during development by injecting pregnant rats with a monoclonal antibody which binds only to these cells. A novel immunization method is being used to produce such antibodies. Antibodies found to bind specifically to SIF cells and/or sympathetic neurons may also be useful in tracing backwards in development to the times and locations at which these differentiation decisions are made.